Compositions and methods for producing clostridial collagenases

ABSTRACT

The present invention provides a method for producing a drug product comprising a combination of highly purified collagenase I and collagenase II from  Clostridium histolyticum . The method utilizes an improved medium for the cultivation of  Clostridium histolyticum  which includes a non-meat-derived (i.e., non-mammalian) peptone or vegetable peptone. The method includes one or more of: (1) reducing glucose content in the meat-free or vegetable-derived media; and (2) increasing the salt concentration in the meat-free or vegetable-derived media. Also provided is a drug product which includes collagenase I and collagenase II at an optimized fixed mass ratio, and which has a purity of greater than at least 95%.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.13/422,939, filed Mar. 16, 2012, which claims the benefit of U.S.Provisional Application No. 61/453,285, filed Mar. 16, 2011, which areincorporated by reference in the entireties.

BACKGROUND OF THE INVENTION

Collagen is the major structural constituent of mammalian organisms andmakes up a large portion of the total protein content of skin and otherparts of the animal body. In humans, it is particularly important in thewound healing process and in the process of natural aging. Various skintraumas such as burns, surgery, infection and accident are oftencharacterized by the erratic accumulation of fibrous tissue rich incollagen and having increased proteoglycan content. In addition to thereplacement of the normal tissue which has been damaged or destroyed,excessive and disfiguring deposits of new tissue sometimes form duringthe healing process. The excess collagen deposition has been attributedto a disturbance in the balance between collagen synthesis and collagendegradation.

Numerous diseases and conditions are associated with excess collagendeposition and the erratic accumulation of fibrous tissue rich incollagen. Such diseases and conditions are collectively referred toherein as “collagen-mediated diseases”. Collagenase, an enzyme that hasthe specific ability to digest collagen, has been used to treat avariety of collagen-mediated diseases. Collagenase formulations have abroad variety of uses in the medical field, including their use astherapeutics.

Collagenase for use in therapy may be obtained from a variety of sourcesincluding mammalian (e.g. human), crustacean (e.g. crab, shrimp),fungal, and bacterial (e.g., from the fermentation of Clostridium,Streptomyces, Pseudomonas, or Vibrio). Collagenase has also beengenetically engineered. One common source of crude collagenase is from abacterial fermentation process, specifically the fermentation ofClostridium histolyticum (“C. histolyticum”). The crude collagenaseobtained from C. histolyticum may be purified using any of a number ofchromatographic techniques.

Collagenases from C. histolyticum, i.e., collagenase class I and classII, are of particular importance, e.g., for dissociation of organ tissuein vitro. Importantly, collagenase digestion of pancreatic tissue ispresently used in the preparation and isolation of human islet cells.However, a number of other different specific cell types have beenisolated from attendant connective tissue, including fat cells fromadipose tissue, hepatocytes from liver, chondrocytes from cartilage,myocytes from heart, and osteoblasts from bone. (Ian Freshney, Cultureof animal cells, A Manual of Basic Technique, Alan R Liss Inc., 1987.)

A practical advantage of using C. histolyticum for the production ofcollagenases is that it can be cultured in large quantities in simpleliquid media, and it regularly produces amounts of proteolytic enzymeswhich are secreted into the culture medium.

As used herein, “meat” culture refers to a culture of cells grown in thepresence of tissue derived from a mammal. “Meat” is not intended toinclude poultry or fish. As used herein, “meat-derived” and“animal-derived” are used interchangeably. Particularly, bovine productshave been used in culture media in the fermentation of C. histolyticum,but they run the risk of contamination by agents which causetransmissible spongiform encephalopathies (TSEs; e.g., prions associatedwith bovine spongiform encephalopathy or “mad cow disease”). A knownmeat culture is the H4 strain of Clostridium histolyticum, which wasdeveloped in Dr. I. Mandl's laboratory at Columbia University in 1956. Afurther meat culture has been derived from the H4 strain named the ABCClostridium histolyticum master cell bank, which has been deposited asATCC 21000.

U.S. Pat. No. 7,811,560 (“the Auxilium '560 patent”), which isincorporated herein by reference in its entirety, discloses methods ofproducing collagenases without using meat-derived media. Using soybeanderived fermentation medium, the methods described therein generatedseparately highly purified collagenase I and II, which were recombinedin a 1:1 ratio to produce a drug product. The Auxilium '560 patent alsodiscloses methods of producing highly purified collagenases usingculture media containing porcine-derived products.

U.S. Patent Publication 2010/0086971 (“the Roche patent publication”),which is also incorporated herein by reference in its entirety,discloses numerous fermentation recipes which are based on vegetablepeptone, including soybean-derived peptone, or vegetable-derived peptoneplus fish gelatin. The Roche patent publication discloses successfulgrowth of Clostridium and collagenase/gelatinase activities in thesemedium conditions, and does not disclose measuring clostripain. However,in another Roche patent U.S. Pat. No. 7,956,167, the inventors disclosethat clostripain activity in the clostridia fermentation broth fordownstream purification is “even more preferred between about 100 and200 U per mg total collagenase”.

One drawback to known methods of fermenting Clostridium for the purposesof isolating collagenase is the production of contaminating proteasessuch as clostripain. Clostripain, a cysteine protease, is believed to bea major cause of collagenase degradation and instability. When proteasesare present in a crude collagenase mixture, researchers must take extraprecautions to neutralize the proteases, including making use ofprotease inhibitors, such as leupeptin, and performing all of thepurification steps in specially designed cold rooms with chilledsolutions. Theoretically, there are two approaches to dealing with theclostripain problem: one approach focuses on the downstream process,i.e., developing a purification method to remove clostripain as early aspossible; the second approach is to develop a method to reduceclostripain production during the fermentation stage.

Thus, the art is in need of improved methods of culturing C.histolyticum. In view of this it is an objective of the invention toprovide alternative methods and compositions for vegetable-derived mediato support the growth of C. histolyticum. and produce collagenase I andII in amounts which can be highly purified from culture supernatant inthe absence of other contaminating proteases such as clostripain.

The inventors have surprisingly found that various components found inprior art fermentation media have a significant impact on the generationof clostripain, which leads to degradation of the desired collagenase Iand II later in the purification process. Specifically, the inventorshave found that either (1) minimizing the glucose content in themeat-free or vegetable-derived media; or (2) increasing the saltconcentration in the meat-free or vegetable-derived media; or acombination of the two approaches, fulfills the above objective.

SUMMARY OF THE INVENTION

The present invention provides a method for producing a collagenasecomposition comprising a combination of highly purified collagenase Iand collagenase II obtained by fermenting C. histolyticum in medium freeof animal material-derived ingredients. In particular, the compositionor drug product obtained by the method is substantiallyclostripain-free, and thus highly stable. By “substantially free” ismeant less than 10 U per mg total collagenase as determined by the assayin example 8 and more preferably less than 5 U/mg, and most preferablyabout 1 U/mg or less; and/or (3) no visible band representingclostripain and/or degraded collagenase on SDS-PAGE gel compared to areference standard.

The present invention also provides fermentation media for Clostridiumhistolyticum, wherein the components of the medium have been optimizedsuch that collagenase I and II are efficiently produced and theproduction of clostripain is inhibited.

The inventors have found that glucose in a meat-free orvegetable-derived medium can support the production of clostripain inthe medium, which can in turn, cause the degradation of the desiredcollagenases. Thus, in one aspect, the invention provides a “lowglucose” meat-free or vegetable-derived medium for the fermentation ofC. histolyticum. A “low glucose” meat-free or vegetable-derived mediumpreferably contains less than about 5 g/L glucose, more preferably lessthan about 1 g/L, even more preferably less than about 0.5 g/L glucose,or can even be glucose-free.

The inventors have also found that using high salt concentrations in thegrowth media can reduce the amount of clostripain produced in culture.Thus, in another aspect, the invention provides a “high salt” meat-freeor vegetable-derived medium for the fermentation of C. histolyticum. By“high salt” is meant greater than about 5 g/L (or 0.5% w/v) total salt,more preferably greater than about 7.5 g/L (or 7.5%) total salt, andmore preferably about 9 g/L (or 9%) or more. It is contemplated that anysalt known to be suitable for use in microbiological fermentation mediamay be used in the current invention. In a preferred embodiment,chloride, phosphate or sulfate salts may be used. In a more preferredembodiment, the salts may be sodium chloride, potassium chloride,monosodium phosphate, disodium phosphate, tribasic sodium phosphate,potassium monophosphate, potassium diphosphate, tripotassium phosphate,calcium chloride, magnesium sulfate or various combinations thereof. Incertain embodiments, potassium diphosphate may be about 0.1-0.3%,potassium phosphate may be about 0.75% to 0.175%, sodium phosphate maybe about 0.2-0.5%, and/or sodium chloride may be about 0.15-0.35%.Preferably, the medium further comprises magnesium sulfate and vitamins,including, riboflavin, niacin, calcium pantothenate, pimelic acid,pyridoxine and thiamine.

In yet another aspect, the invention provides a collagenase compositionor product obtained through the process of the invention which maycontain a mass ratio of collagenase I and collagenase II which ismodified or optimized to produce a desired or even a maximal synergisticeffect. In certain embodiments, the collagenase I to collagenase II massratio is about 0.5 to 1.5, more preferably 0.6 to 1.3, even morepreferably 0.8 to 1.2, and most preferably, 1 to 1.

In still other aspects, the invention provides pharmaceuticalformulations and methods for treating patients suffering from acollagen-mediated disease using a collagenase composition of theinvention.

Yet a further aspect of the invention is a method for cultivatingClostridium histolyticum in a meat-free or vegetable-derived medium,wherein the medium is “low glucose” or “high salt” or both.

The currently claimed methods result in the surprising reduction in theamount of clostripain produced in cultures of Clostridium histolyticum,thus eliminating the need for adding protease inhibitors such asleupeptin to reagents and allowing researchers to conduct allpurification steps at ambient temperature. The foregoing and otherobjects, features and advantages of the invention will be apparent fromthe following more particular description of preferred embodiments ofthe invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photomicrograph of an SDS-PAGE gel showing the results of anexperiment comparing media with and without glucose or salt. The doubleband indicated by the upper arrow is collagenase I and II. The bandindicated by the lower arrow is clostripain. The protein samples of eachlane are identified as follows:

Lane 1: Marker

Lane 2: Marker

Lane 3: M #1 seed culture

Lane 4: M #3

Lane 5: M #3+additional yeast extract

Lane 6: M #3−salt

Lane 7: M #3+glucose

Lane 8: M #3−salt/+glucose

Lane 9: M #3

Lane 10: M #3+additional yeast extract

Lane 11: M #3−salt

Lane 12: M #3+glucose

Lane 13: M #3−salt/+glucose

Lane 14: Marker

FIG. 2 is a photomicrograph of an SDS-PAGE gel showing the results of anexperiment using phytone and Veggietone media with and without glucose.The double band indicated by the upper arrow is collagenase I and II;the band indicated by the lower arrow is clostripain. The proteinsamples of each lane are identified as follows:

Lane 1: Marker

Lane 2: M #1

Lane 3: M#3 (phytone)

Lane 4: M #4 (Veggietones)

Lane 5: M #3+glucose

Lane 6: M #4+glucose

Lane 7: M #3 (phytone)

Lane 8: M #4+glucose

Lane 9: M #3+glucose

Lane 10: M #4+glucose

Lane 11 Marker

Lane 12: Marker

FIG. 3 is a photomicrograph of an SDS-PAGE gel showing the results of anexperiment using various Veggietone media formulations with and withoutsalt. The double bands indicated by the upper arrow are the collagenasesI and II; the band indicated by the lower arrow is clostripain. Theprotein samples of each lane are identified as follows:

Lane 1: Marker

Lane 2: seed culture

Lane 3: 5% oxoid VG 0100/1 mM CaCl₂

Lane 4: M #4

Lane 5: M #4−salt+1 mM CaCl₂

Lane 6: M #3

Lane 7: M #4

Lane 8: M #4−salt+1 mM CaCl₂

Lane 9: M #3

Lane 10: Marker

Lane 11: Marker

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a method for producing collagenases by fermentingC. histolyticum in a meat-free or vegetable-derived medium, wherein theculture supernatant is substantially clostripain-free. The collagenasesso produced can be isolated, purified, and combined to provide acomposition comprising a mixture of collagenase I and collagenase II inan optimized fixed mass ratio which is substantially clostripain-free.It is understood that the terms “drug substance”, “drug product” or“collagenase composition” can be used interchangeably.

In one embodiment of the invention, a fermentation process is described.The crude collagenase obtained from fermentation of C. histolyticum maybe purified by a variety of methods known to those skilled in the art,including dye ligand affinity chromatography, heparin affinitychromatography, ammonium sulfate precipitation, hydroxylapatitechromatography, size exclusion chromatography, ion exchangechromatography, and/or metal chelation chromatography. Additionally,purification methods for collagenases are known, such as, for example,those described in the Auxilium '560 patent.

Both collagenase I and collagenase II are metalloproteases and requiretightly bound zinc and loosely bound calcium for their activity (EddieL. Angleton and H. E. Van Wart, Biochemistry 1988, 27, 7406-7412). Bothcollagenases have broad specificity toward all types of collagen(Steinbrink, D; Bond, M and Van Wart, H; (1985), JBC, 260 p2771-2776).Collagenase I and Collagenase II digest collagen by hydrolyzing thetriple-helical region of collagen under physiological conditions(Steinbrink, D; Bond, M and Van Wart, H; (1985), JBC, 260 p2771-2776).Even though each collagenase shows different specificity (e.g. each havea different preferred target amino sequence for cleavage), together,they have synergistic activity toward collagen [Mandl, I., (1964),Biochemistry, 3: p. 1737-1741; Vos-Scheperkeuter, G H, (1997), CellTransplantation, 6: p. 403-412]. Collagenase II has a higher activitytowards all kinds of synthetic peptide substrates than collagenase I asreported for class II and class I collagenase in the literatures. [Bond,M. D. (1984), Biochemistry, 23: p. 3085-3091. Hesse, F, (1995),Transplantation Proceedings, 27: p. 3287-3289].

A “peptone” is understood as being a mixture of any of variouswater-soluble compounds that form as intermediates during hydrolysis ofproteins to amino acids. A peptone is frequently obtained by enzymaticdigestion or acid hydrolysis of natural products, such as animaltissues, milk, plants or microbial cultures. In nutrient media forgrowing microorganisms such as bacteria and fungi, peptones and gelatinscan serve as an organic source, e.g., for carbon and/or nitrogen.

Frequently, the protein source for the production of a peptone is awaste form arising during the production of meat and dairy produce.However, a variety of peptones are available from plant sources.Depending on the source material and any processing thereof (such aspurification to a certain degree of the proteinaceous components of thesource) before hydrolytic treatment, a number of compounds other thanpeptides or amino acids can be part of a peptone.

The term “gelatin” refers to a solid or semi-solid substance extractedfrom collagen-containing connective tissue of multicellular animals(metazoans). Collagen proteins, herein collectively referred to as“collagen”, have a structural function in the extracellular matrix.Collagen proteins are known to occur not only in higher animals such asmammals but even in very primitive sea sponges.

Gelatin is an irreversibly hydrolyzed form of collagen proteins and isproduced by partial hydrolysis of collagen extracted from skins, bones,cartilage, connective tissues, organs, and intestines. The chemicalcomposition of gelatin is similar to that of collagen. Gelatin is formedwhen the natural molecular bonds between individual collagen strands arebroken down into a form that rearranges more easily. Thus, gelatin meltswhen heated and solidifies when cooled again.

In the biotech industry, a number of enzymes for use in pharmaceuticalprocesses are produced in large-scale fermentation processes. Examplesinclude C. histolyticum collagenase enzymes for the dissociation oforgan tissue and the subsequent isolation of target cells from thedissociated organ tissue. Because the microbial culture producing theenzymes takes up components from the growth media, it is desirable todevelop media with peptones and extracts free of mammalian pathogenicagents. There is a particular safety-related concern regarding prionsand bovine spongiform encephalopathy (BSE).

The drug substance of the invention, for injectable collagenase,consists of two microbial collagenases, referred to as Collagenase ABC Iand Collagenase ABC II. It is understood that the terms “Collagenase I”,“ABC I”, and “collagenase ABC I” mean the same and can be usedinterchangeably. Similarly, the terms “Collagenase II”, “ABC II”, and“collagenase ABC II” refer to the same enzyme and can also be usedinterchangeably. These collagenases are secreted by bacterial cells.They are isolated and purified from Clostridium histolyticum culturesupernatant by chromatographic methods. Both collagenases are specialproteases and share the same EC number (E.C 3.4.24.3).

Collagenase ABC I has a single polypeptide chain consisting ofapproximately 1000 amino acids with a molecular weight of 115 kDa.Collagenase ABC II has also a single polypeptide chain consisting ofabout 1000 amino acids with a molecular weight of 110 kDa.

Even though the literature indicates that there are sequence homologiesin regions of collagenase ABC I and ABC II, the two polypeptides do notseem to be immunologically cross reactive as indicated by the westernblot analysis.

A first step in the method of the present invention may be thepreparation of meat-free or vegetable-derived clostridia cell bank.Preferably, the Clostridium histolyticum is removed from a meat mediumand resuspended in a medium containing a vegetable peptone andoptionally yeast extract. An exemplary, albeit non-limiting, example ofthe steps involved in the preparation of an animal material-freeclostridia cell bank is as follows:

Step 1 Starting cells: Clostridium histolyticum ATCC 21000, strain 004in meat-derived medium; Step 2 Inoculate 1 mL of step 1 into 300 mL ofmedia #1; Step 3 Incubate step 2 for 24 hours at 37° C. (1^(st)culture); Step 4 Transfer 3 mL of step 3 (1^(st) culture) to 1000 mL ofmedia #1; Step 5 Incubate step 4 for 16 hours at 37° C. (2^(nd)culture); Step 6 Centrifuge the 2^(nd) culture; Step 7 Re-suspend thepellet with the 5 mL of media #1 and 5 mL of 20% glycerol; Step 8 Freezethe aliquot of cells gradually; Step 9 Store the aliquot at −80° C.

Once an animal material-free cell bank is established, the clostridiacan be grown or fermented in various media, preferably vegetable-derivedmedium. The medium can optionally contain yeast extract. Exemplary,albeit non-limiting examples of such media are media designated M#1-#4below. In addition, an exemplary, albeit non-limiting example of thesteps of the fermentation process are as follows:

Step 1 Starting cells: Animal material free clostridia cell bank Step 2Inoculate 1 mL of step 1 into the 300 mL of media #1; Step 3 Incubatestep 2 for 16 to 24 hours at 37° C. (1^(st) culture); Step 4 Transfer 10mL of step 3 (1^(st) culture) and 10 mL Vitamin/Mg solution to 1000 mLof media # 3, or 4 respectively; Step 5 Incubate step 4 for about 22hours at 37° C. (2^(nd) culture); Step 6 2^(nd) culture for downstreamisolation and purification.

After preparation of “2^(nd) culture”, the collagenase I and collagenaseII can be isolated and purified using any method capable of producingeach enzyme separately to at least 95% purity. The method may combineone or more of the steps of ammonium sulfate precipitation, dialysis,hydroxylapatite (HA) chromatography, gel filtration and ion-exchange,preferably in that order. The gel filtration is preferably G75 gelfiltration. The ion-exchange is preferably anion-exchange: Q-Sepharosechromatography. In addition, when the clostridia have been preferablycultured in medium containing less glucose and more salt, the use ofprotease inhibitors such as leupeptin is not required. An exemplary,albeit non-limiting example of the isolation and purification steps forcollagenase I and collagenase II are as follows:

Stages of Product Operations Fermentation broth Centrifugation or 1.0 μmfiltration; Clarified fermentation Add ammonium sulfate (590 g/liter)and followed broth by centrifugation; Crude Collagenase Dissolve CrudeCollagenase Precipitate by adding Precipitate purified water; Crudecollagenase Note: can be stored at −20° C.; solution Dialyze crudecollagenase solution against purified water overnight with 10 kDa poresize dialysis membrane; Dialyzed crude Clarify the dialyzed crudecollagenase solution collagenase with either centrifugation orfiltration or the combination of both; Clarified solution Add potassiumphosphate buffer, pH 6.7 to a final concentration of 0.1M; Collagenasein Load Collagenase solution to a hydroxylapatite phosphate buffer (HA)column and elute the column using a gradient elution of increasingpotassium phosphate concentration at ambient temperature (20° C.);Collagenase HA Concentrate the eluate with ultrafiltration (30 eluatekDa of pore size); Concentrated Load the concentrate onto a G75 gelfiltration collagenase column at ambient temperature (20° C.) and elutewith 20 mM Tris/150 mM NaCl; Collagenase G75 Dialyze the eluate againsta buffer (10 mM Tris, eluate 3 mM calcium chloride (CaCl₂), pH 8.0)overnight; Dialyzed G75 eluate Load the dialyzed eluate on to aQ-Sepharose anion-exchange column at ambient temperature (20° C.) andelute using a gradient of 10 mM Tris HCl, 3 mM CaCl₂, pH 8.0 Buffer and10 mM Tris HCl, 3 mM CaCl₂, 1M NaCl, pH 8.0 Buffer; Collagenase class IStore them separately at −20° C. fraction Collagenase class II fraction

According to the invention, the peptone in the nutrient composition isan animal material-free derived peptone. In a preferred embodiment ofthe invention, the peptone is a plant product. Even more preferred, thepeptone is from a plant source selected from the group consisting of soybean, broad bean, pea, potato, and a mixture thereof. The peptone may beselected from the group consisting of Oxoid VG100 Vegetable peptone No.1 from pea (VG100), Oxoid VG200 Vegetable peptone phosphate broth fromPea (VG200), Merck TSB CASO-Bouillion animal-free (TSB), Invitrogen Soybean peptone No 110 papainic digest (SP6), Fluka Broad bean peptone(BP), Organotechnie Plant peptone E1 from potato (E1P), BBL Phytone™peptone and BD Difco Select Phytone™.

Regarding configuration of the nutrient medium and peptone concentrationin particular, the composition according to the invention may comprisetwo or more different peptones, whereby the aggregate concentration ofthe plant peptones in the composition is between about 2% and about 15%weight by volume. Even more preferred, the aggregate concentration ofthe plant peptones in the composition is between about 5% and about 12%weight by volume, preferably about 10% weight by volume.

In a preferred embodiment of the invention, a single type of peptone ispresent in the nutrient composition of the invention, whereby thepeptone is selected from the group consisting of BP, E1P, Soy beanpeptone E110, VG100, and VG200, and whereby the concentration of thepeptone in the composition is about 5% weight by volume. In yet anothervery much preferred embodiment of the invention, a single type ofpeptone is present in the nutrient composition of the invention, wherebythe peptone is BBL phytone peptone or Difco Select Phytone™ UF, andwhereby the concentration of the peptone in the composition is about10-13% weight by volume.

In another preferred embodiment, the nutrient composition may contain0.5-5% yeast extract, more preferably about 1-4%, and most preferablyabout 1.5-2.5%. Yeast extract is available from a variety of suppliers,including Cole Parmer (Vernon Hills, Ill.) and Fisher Scientific(Pittsburgh, Pa.).

In yet a preferred embodiment of the invention, the pH of the media isbetween pH 7 and pH 8. Even more preferred is a pH between about pH 7.2and about pH 7.7, most preferably about 7.4.

In a further preferred embodiment, the media according to the inventionare sterilized, that is to say the composition is made free of anyself-replicating organism. Sterilization can be achieved by standardmethods known to the skilled person, e.g., by heat treatment such asautoclaving.

The media composition according to the invention is particularly usefulfor cultivating Clostridium histolyticum bacteria. A preferredembodiment of the invention is therefore a composition according to theinvention additionally comprising an inoculum of Clostridiumhistolyticum bacteria in a media composition of the invention.

In another embodiment, the present invention provides a drug substance,wherein the drug substance has at least one specification selected fromtable A below:

TABLE A Specification Test ABC-I ABC-II Appearance Clear colorless andessentially free from particulate matter Endotoxin <10 EU/mL Identity(and purity) Major Major by SDS-PAGE collagenase collagenase (Reducedconditions, band between band between Coomasie) 98-188 97-200 kDa ≥ 95%kDa ≥ 95% SRC assay (ABC -I) 1967-3327 NA SRC units/mg GPA assay (ABC-IINA 81934-119522 GPA units/mg Analysis of Proteins ≥98% main peak; ≤2%aggregates by area HPLC System (Aggregation by size exclusionchromatography) Identity and purity Major peak (ABC I or ABC II), ≥95%by area; by reverse phase Retention times of ABC-I and liquid ABC-IIwithin 5% of reference chromatography) Clostripain assay ≤1 U/mg  (BAEEassay) Bioburden  <1 cfu/mL

Such drug product of the invention is useful for the treatment ofcollagen-mediated disease. Examples of such collagen mediated-diseasesthat may be treated by the compositions and methods of the inventioninclude but are not limited to: Dupuytren's disease; Peyronie's disease;frozen shoulder (adhesive capsulitis), keloids; tennis elbow (lateralepicondylitis); scarred tendon; glaucoma; herniated discs; adjunct tovitrectomy; hypertrophic scars; depressed scars such as those resultingfrom inflammatory acne; post-surgical adhesions; acne vulgaris; lipomas,and disfiguring conditions such as wrinkling, cellulite formation andneoplastic fibrosis. U.S. Pat. No. 6,086,872, incorporated herein byreference in its entirety, disclose the use of collagenase preparationsin the treatment of Dupuytren's disease. U.S. Pat. No. 6,022,539,incorporated herein by reference in its entirety, discloses the use ofcollagenase preparations in the treatment of Peyronie's disease. U.S.patent application Ser. No. 12/266,090, incorporated herein by referencein its entirety, discloses the treatment of adhesive capsulitis withpurified preparations of collagenase. U.S. patent application Ser. No.11/703,269, incorporated herein by reference in its entirety, disclosesthe treatment of cellulite with purified preparations of collagenase.U.S. Pat. Nos. 7,824,673 and 6,958,150, incorporated herein by referencein their entirety, discloses the treatment of adipose tissue withpurified preparations of collagenase. U.S. Pat. No. 7,854,929,incorporated herein in its entirety, discloses the treatment of lateralepicondylitis using purified preparations of collagenase.

In addition to its use in treating collagen-mediated diseases, thecomposition of the invention is also useful for the dissociation oftissue into individual cells and cell clusters as is useful in a widevariety of laboratory, diagnostic and therapeutic applications. Theseapplications involve the isolation of many types of cells for varioususes, including microvascular endothelial cells for small diametersynthetic vascular graft seeding, hepatocytes for gene therapy, drugtoxicology screening and extracorporeal liver assist devices,chondrocytes for cartilage regeneration, and islets of Langerhans forthe treatment of insulin-dependent diabetes mellitus. Enzyme treatmentworks to fragment extracellular matrix proteins and proteins whichmaintain cell-to-cell contact. Since collagen is the principle proteincomponent of tissue ultrastructure, the enzyme collagenase has beenfrequently used to accomplish the desired tissue disintegration. Ingeneral, the composition of the present invention is useful for anyapplication where the removal of cells or the modification of anextracellular matrix, are desired.

Collagenase compositions of the invention may also be prepared by mixingeither a specific number of activity units or specific masses of thepreferably purified enzymes. Collagenase activity can be measured by theenzyme's ability to hydrolyze either synthetic peptide or collagensubstrate. Those skilled in the art will recognize that enzyme assaysother than those disclosed herein may also be used to define and preparefunctionally equivalent enzyme compositions.

Another aspect of the present invention is the reproducible optimizationof the mass ratio of collagenase I to collagenase II in the compositionof the invention. The reproducibility of the ratio of collagenase I tocollagenase II has previously been a challenge because of severalfactors. Commercial fermentation of Clostridium can result in a 1 to 2ratio of collagenase I and collagenase II. By separately purifyingcollagenase I and collagenase II, and recombining them at an optimizedfixed mass ratio, the present invention maximizes the synergisticactivity provided by the two different collagenases resulting insuperior therapeutic benefit.

The invention also provides pharmaceutical formulations of thecompositions of the invention. The pharmaceutical formulations of thepresent invention comprise a therapeutically effective amount of acollagenase composition of the present invention formulated togetherwith one or more pharmaceutically acceptable carriers or excipients.

As used herein, the term “pharmaceutically acceptable carrier orexcipient” means a non-toxic, inert solid, semi-solid or liquid filler,diluent, encapsulating material or formulation auxiliary of any type.Some examples of materials which can serve as pharmaceuticallyacceptable carriers are sugars such as lactose, glucose and sucrose;starches such as corn starch and potato starch; cellulose and itsderivatives such as sodium carboxymethyl cellulose, ethyl cellulose andcellulose acetate; powdered tragacanth; malt; gelatin; talc; glycolssuch as propylene glycol; esters such as ethyl oleate and ethyl laurate;agar; buffering agents such as magnesium hydroxide and aluminumhydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer'ssolution; ethyl alcohol, and phosphate buffer solutions, as well asother non-toxic compatible lubricants such as sodium lauryl sulfate andmagnesium stearate, as well as coloring agents, releasing agents,coating agents, perfuming agents, preservatives and antioxidants canalso be present in the composition, according to the judgment of theformulator.

The pharmaceutical compositions of this invention may be administeredparenterally, topically, or via an implanted reservoir. The termparenteral as used herein includes subcutaneous, intracutaneous,intravenous, intramuscular, intraarticular, intraarterial,intrasynovial, intrasternal, intrathecal, intralesional and intracranialinjection or infusion techniques. In a preferred embodiment, thecomposition is injected into the disfiguring tissue. In the case ofPeyronie's or Duputyren's diseases or adhesive capsulitis, thecomposition is injected into the cord or plaque. The term “localadministration” is defined herein to embrace such direct injection. U.S.Pat. Nos. 5,393,792 and 5,422,103, incorporated herein by reference intheir entirety, disclose compositions of collagenase used for topicaladministration to burn sites.

Furthermore, particularly good results can be obtained by immobilizingthe site of injection after administration. For example, the site ofadministration can be immobilized for 4 or more hours.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions, may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use. The sterile solutions may also be lyophilized forlater use.

Dosage forms for topical or transdermal administration of a compound ofthis invention include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants or patches. The active componentis admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to the compounds of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants suchas chlorofluorohydrocarbons.

Transdermal patches have the added advantage of providing controlleddelivery of a compound to the body. Such dosage forms can be made bydissolving or dispensing the compound in the proper medium. Absorptionenhancers can also be used to increase the flux of the compound acrossthe skin. The rate can be controlled by either providing a ratecontrolling membrane or by dispersing the compound in a polymer matrixor gel.

In one preferred embodiment, the drug substance of the invention is alyophilized injectable composition formulated with lactose. A preferredcollagenase composition comprising a mixture of collagenase I andcollagenase II has a specific activity of at least about 700 SRCunits/mg, such as at least about 1000 SRC units/mg, more preferably atleast about 1500 SRC units/mg. One SRC unit will solubilize rat tailcollagen into ninhydrin reaction material equivalent to 1 nanomole ofleucine per minute, at 25° C., pH 7.4.

Collagenase has been described in ABC units as well. This potency assayof collagenase is based on the digestion of undenatured collagen (frombovine tendon) at pH 7.2 and 37 degrees C. for 20-24 hours. The numberof peptide bonds cleaved are measured by reaction with ninhydrin. Aminogroups released by a trypsin digestion control are subtracted. One netABC unit of collagenase will solubilize ninhydrin reactive materialequivalent to 1.09 nanomoles of leucine per minute. One SRC unit equalapproximate 6.3 ABC unit or 18.5 GPA unit.

In another embodiment, each milligram of injection collagenasepreferably has approximately 2800 SRC units measured with a potencyassay using soluble rat tail collagen as the substrate, and 51000 unitsmeasured with a potency assay using a synthetic substrate, pzGPGGPA.

In another preferred embodiment, the collagenase composition of theinvention is a lyophilized injectable composition formulated withSucrose, Tris at a pH level of about 8.0. Most preferably, 1.0 mg of thedrug substance of the invention is formulated in 60 mM Sucrose, 10 mMTris, at a pH of about 8.0 (this equates to 20.5 mg/mL of sucrose and1.21 mg/mL of Tris in the formulation buffer). Examples of some of theformulations include, but not limited to: for a 0.9 mg of the drugsubstance dose, 18.5 mg of sucrose and 1.1 mg of Tris are added in eachvial, where the targeting a vial fill volume is 0.9 mL; and for a 0.58mg of the drug substance dose, 12.0 mg sucrose (multicompendial) and 0.7mg of Tris (multicompendial).

In accordance with the invention, methods are provided for treatingcollagen-mediated diseases comprising the step of administering to apatient in need thereof, a therapeutically effective amount of acomposition of the invention, or a therapeutically effective amount of apharmaceutical formulation of the invention. By a “therapeuticallyeffective amount” of a compound of the invention is meant an amount ofthe compound which confers a therapeutic effect on the treated subject,at a reasonable benefit/risk ratio applicable to any medical treatment.

The therapeutic effect may be objective (i.e., measurable by some testor marker) or subjective (i.e., subject gives an indication of or feelsan effect). Effective doses will also vary depending on route ofadministration, as well as the possibility of co-usage with otheragents. It will be understood, however, that the total daily usage ofthe compositions of the present invention will be decided by theattending physician within the scope of sound medical judgment. Thespecific therapeutically effective dose level for any particular patientwill depend upon a variety of factors including the disorder beingtreated and the severity of the disorder; the activity of the specificcompound employed; the specific composition employed; the age, bodyweight, general health, sex and diet of the patient; the time ofadministration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or contemporaneously with the specific compound employed;and like factors well known in the medical arts.

In yet more detail, the present invention is described by the followingitems which represent preferred embodiments thereof.

-   -   1. A method for preparing a drug product comprising isolated and        purified collagenase I and collagenase II having the sequence of        Clostridium histolyticum collagenase I and collagenase II, said        method including the steps of:        -   a) fermenting Clostridium histolyticum in an animal-derived            material-free medium to obtain a crude collagenase mixture,            wherein said medium is treated in one or more of the            following ways: (1) decreasing the glucose content in the            animal-derived material-free medium; and (2) increasing the            salt concentration in the animal-derived material-free            medium;        -   b) purifying collagenase I and collagenase II separately            from the crude collagenase mixture; and        -   c) recombining the purified collagenase I and collagenase II            at an optimized fixed mass ratio;        -   wherein said drug product is at least 95% pure and            substantially free of any contaminating proteases.    -   2. The method of Item 1, wherein the animal-derived        material-free media comprises vegetable-derived peptone.    -   3. The method of Item 1, wherein the animal-derived        material-free media comprises soybean-derived peptone.    -   4. The method of Item 1, wherein the optimized fixed mass ratio        of collagenase I to collagenase II is 0.5 to 1.5.    -   5. The method of Item 1, wherein the optimized fixed mass ratio        of collagenase I to collagenase II is 1.    -   6. The method of Item 1, wherein the collagenases are 95% pure.    -   7. The method of Item 6, wherein the collagenases are 98% pure.    -   8. The method of Item 1, wherein the purity is determined by one        or more of the following: sodium dodecyl sulfate polyacrylamide        gel electrophoresis (SDS-PAGE), high performance liquid        chromatography (HPLC), reverse-phase HPLC, or enzymatic assays.    -   9. The method of Item 1, wherein the drug product is essentially        clostripain-free.    -   10. The method of Item 1, wherein step (b) comprises:        -   i. ammonium sulfate precipitation;        -   ii. dialysis;        -   iii. hydroxylapatite (HA) chromatography;        -   iv. gel filtration chromatography; and        -   v. anion exchange chromatography.    -   11. The method of Item 11, wherein steps (i), (ii), (iii), (iv)        and (v) are performed in order.    -   12. The method of Item 11, wherein the column used for        chromatography is a hydroxylapatite (HA) column.    -   13. The method of Item 11, wherein the gel filtration is G75 gel        filtration.    -   14. The method of Item 11, wherein the anion-exchange is        Q-Sepharose anion-exchange.    -   15. The method of Item 1, wherein the purification is performed        in the absence of leupeptin.    -   16. The method of Item 1, further including preparing an animal        material-free clostridia cell bank.    -   17. The method of Item 1, wherein the glucose content in the        animal material-free medium is less than about 5 g/L.    -   18. The method of Item 1, wherein the glucose content in the        animal material-free medium is less than about 2 g/L.    -   19. The method of Item 1, wherein the glucose content in the        animal material-free medium is essentially zero.    -   20. The method of Item 1, wherein the salt content in the animal        material-free medium is greater than about 5 g/L.    -   21. The method of Item 1, wherein the salt content in the animal        material-free medium is greater than about 7.5 g/L.    -   22. The method of Item 1, wherein the salt content in the animal        material-free medium is greater than about 9 g/L.    -   23. A drug product prepared by the method of any of Items 1-23.    -   24. A medium for the fermentation of Clostridium histolyticum        including:        -   a. Phytone or veggitone;        -   b. yeast extract; and        -   c. less than about 5 g/L glucose;        -   wherein the pH of said medium is 7.2-7.7.    -   25. A medium for the fermentation of Clostridium histolyticum        comprising:        -   a. phytone or veggitone        -   b. yeast extract; and        -   c. greater than about 5 g/L salt;        -   wherein the pH of said medium is 7.2-7.7.    -   26. The medium of Item 26, wherein the salts are selected from        the group consisting of potassium phosphate, sodium phosphate,        sodium chloride, and magnesium sulfate.    -   27. The medium of any one of Items 25-27, further comprising one        or more vitamins selected from the group consisting of ferrous        sulfate, riboflavin, niacin, calcium pantothenate, pimelic acid,        pyridoxine hydrochloride and thiamine hydrochloride.    -   28. The medium of Item 25, wherein the pH of said medium is 7.4.    -   29. A method for treating a patient suffering from a        collagen-mediated condition or disease, comprising administering        to said patient a therapeutically effective amount of the drug        product of Item 24.    -   30. The method of Item 30, wherein said condition or disease is        Dupuytren's disease, Peyronie's disease, frozen shoulder        (adhesive capsulitis), tennis elbow (lateral epicondylitis),        keloids, scarred tendon, glaucoma, herniated discs, adjunct to        vitrectomy, hypertrophic scars, depressed scars, post-surgical        adhesions, acne vulgaris, lipomas, wrinkling, cellulite        formation or neoplastic fibrosis.

All references cited herein, whether in print, electronic, computerreadable storage media or other form, are expressly incorporated byreference in their entirety, including but not limited to, abstracts,articles, journals, publications, texts, treatises, internet web sites,databases, patents, and patent publications.

EXAMPLES

The compositions and processes of the present invention will be betterunderstood in connection with the following examples, which are intendedas an illustration only and not limiting of the scope of the invention.Various changes and modifications to the disclosed embodiments will beapparent to those skilled in the art and such changes and modificationsincluding, without limitation, those relating to the processes,formulations and/or methods of the invention may be made withoutdeparting from the spirit of the invention and the scope of the appendedclaims.

Example 1 Preparation of an Animal Material Free Cell Bank

The starter cell culture was Clostridium histolyticum ATCC 21000, strain004, which was originally created with bovine-derived materials. Thecells were first grown in animal material free medium (M #1, Table 1).Briefly, the recipe includes: phytone, 51.5 g, yeast extract 8.5 g, 1000mL water. The pH was adjusted to 7.30 with NaOH, and sterilized at 121°C. for 20 min. 1 mL of the starting material was then inoculated into300 mL of media #1 and incubated for 24 hours at 37° C. (1^(st)culture). 3 mL of the 1^(st) culture was transferred to 1000 mL of media#1 and incubated for 16 hours (2^(nd) culture). The 2^(nd) culture wasthen centrifuged aseptically. The pellet was re-suspended with 5 mLmedia #1 and 5 mL 20% glycerol. The aliquots of cell suspension werefrozen gradually and stored at −80° C.

Example 2 Fermentation Process

Clostridium histolyticum ATCC 21000, strain 004 was inoculated into thestarting culture with medium #1 or #2 and incubated at 37° C. for 16 hr.10 mL of the starting culture (M#1 or #2) and 10 mL Mg/vitamin solution(Example 3) was then transferred to each liter of M#3 or #4 or anyvariation to either M#3 or M#4 specified in the examples and incubatedfor 22 hours. Clostridium histolyticum grew well with the OD₆₀₀ reaching>2.5.

TABLE 1 Media recipes and preparation M #1 M #2 M #3 M #4 Phytone 15.45g 103 g Veggitone 15.45 g 103 g Yeast extract 2.55 g 2.55 g 17 g 17 gKH2PO4 1.92 g 1.92 g K2HPO4 1.25 g 1.25 g Na2HPO4 3.5 g 3.5 g NaCl 2.5 g2.5 g vol of water 0.3 L 0.3 L 1 L 1 L

Example 3 Preparation of MgSO₄/VF Solution

The solution was prepared separately, by dissolving 8 g MgSO₄, 1.2 gferrous sulfate, 0.05 g riboflavin, 0.1 g Niacin, 0.1 g Calciumpantothenate, 0.1 g pimelic acid, 0.1 g pyridoxine, and 0.1 g thiaminein 1100 mL water. The solution was sterilized with 0.22 um filtration.

Example 4 Fermentation Medium Study: Effect of Glucose and Salt

SDS-PAGE Analysis

The M#3 medium was modified with additional components as described inTable 2. The fermentation broths were analyzed by SDS-PAGE.

TABLE 2 Formation Modifications Remove Additional Medium modificationGlucose salt Yeast extract M #3 No No No M #3 + additional yeast extractNo No 33 g/L M #3 − salt No Yes No M #3 + glucose 10 g/L No No M #3 −salt + glucose 10 g/L Yes No Salt: KH2PO4, K2HPO4, Na2HPO4, NaCl asdescribed in Table 1.

FIG. 1 shows the results of an SDS-PAGE analysis of culture supernatantsgrown in the presence or absence of salt and the presence or absence ofglucose. FIG. 1 shows that fermentation with glucose (lane 7) increasesthe production of clostripain (the 43 kDa band) when compared tofermentation without glucose (lane 4).

FIG. 1 also shows that clostripain production increases drastically whenfermentation occurs in the absence of salt (lanes 6, 8, 11 and 13)compared to fermentation in the presence of salt (lanes 4 and 9; noticethe more prominent 43 kDa band).

Casein Assay

It was also shown through a caseinase assay that glucose in mediaincreases the production of proteolytic enzymes that contribute to thethe degradation of collagenase. The caseinase assay is commonly used tomeasure general proteolytic activity by assaying the proteolyticdegradation of the substrate casein. The fermentation broths from M #3with and without glucose were processed with ammonium sulfate and theprecipitates were analyzed for caseinase activity (exp: 1325). For M #3with glucose, the precipitate showed activity of 80.0 unit per unit ofcell density (OD600). The precipitate from M #3 without glucose, showedactivity of 60.5 unit per unit of cell density. Thus, the glucose growthcondition displayed 32% more caseinase activity. Higher caseinaseactivity is consistent with the presence of clostripain and maycontribute to increased degradation of collagenase.

Example 5 Fermentation Medium Study: Veggietone Vs Phytone with andwithout Glucose

Cultures of C. histolyticum were grown in phytone-based media andVeggietone-based media, with and without added glucose. FIG. 2 shows theresults of an SDS-PAGE analysis of the protein isolated from thecultures.

A comparison of proteins derived from cells grown in phytone andVeggietone (lane 7 and 8) revealed that Veggietone produced much lessclostripain. Additionally, a comparison of proteins derived from cellsgrown in phytone with glucose (lanes 5 and 9) and phytone withoutglucose (lanes 3 and 7) revealed that the use of media with glucosedrastically increased the production of clostripain compared to mediawithout glucose. In contrast, Veggietone media without glucose did notdisplay the same effect. A comparison of proteins derived from cellsgrown in Veggietone with glucose (lanes 6 and 10) and Veggietone withoutglucose (lanes 4 and 8) indicates that the addition of glucose toVeggietone, did not drastically increase clostripain production.However, this may be explained by the presence of salt in the Veggietonemedia. (See example 6).

Moreover, the Veggietone-derived protein sample revealed a consistentphenomenon: some collagenase class I precursor did not fully convert tomature collagenase class I. The conversion process continued afterammonium sulfate precipitation, and completed before the ion exchangecolumn.

Yield: approximately 20 mg/L broth for either ABC I or II.

Example 6 Fermentation Medium Study: Veggietone and Salt

Clostripain levels were measured in protein samples derived from C.histolyticum cultures grown in media prepared with and without salt.Cells were grown in Veggietone media with salt, Veggietone media withoutsalt and with CaCl₂, phytone growth media or 5% Veggietone media withCaCl₂ (disclosed in U.S. patent application Ser. No. 12/478,306). FIG. 3reveals that clostripain (43 kDa band) production was drasticallyreduced in cultures grown in Veggietone with salt (lanes 4 and 7)compared to growth in Veggietone without salt (lanes 5 and 8). Proteinsamples derived from cultures grown in phytone (lanes 6 and 9) or 5%Veggietone and 1 mM CaCl2 (lane 3) also displayed increased productionof clostripain compared to Veggietone with salt.

Example 7 The Isolation and Purification Process

After fermentation in medium #4, the Clostridia cells were removed bycentrifugation or 1 μm filtration. Ammonium sulfate (590 g/liter) wasadded to the clarified fermentation solution to precipitate out thecollagenases. The Collagenase Precipitation was recovered bycentrifugation, reconstituted with purified diH₂O and dialyzed againstpurified diH₂O for at least 14 hours.

The dialyzed, Crude Collagenase solution was centrifuged at 20,000 g for15 minutes at 2-8° C., the supernatant was filtered through a 0.22 μmnitrocellulose membrane and the filtered solution was adjusted to 0.1Mpotassium phosphate (KPi) by adding 0.4M KPi, pH 6.7 Buffer. Thisdiluted Collagenase solution was then run through a 185 mLhydroxylapatite (HA) column and the collagenase was eluted using agradient of increasing potassium phosphate concentration at ambienttemperature (20° C.). The hydroxylapatite column captured collagenasewhile allowing all the non-collagenase material to go through theflow-through fraction. In addition, the gradient elution also helped topartially remove DNA impurities.

The resulting collagenase fractions were pooled, concentrated with 30kDa ultrafiltration, and loaded onto a G75 gel filtration column atambient temperature (20° C.). The collagenase was eluted with 20 mMTris/150 mM NaCl. The collagenase fractions were pooled and dialyzedagainst buffer A (10 mM Tris, 3 mM calcium chloride (CaCl₂), pH 8.0. Thedialyzed collagenase pool was then filtered through a 0.22 μmnitrocellulose filter.

The filtered collagenase in Tris Buffer solution was then run through aQ-Sepharose anion-exchange column at ambient temperature (20° C.) andeluted using a gradient of 10 mM Tris HCl, 3 mM CaCl₂, pH 8.0 Buffer and10 mM Tris HCl, 3 mM CaCl₂, 1M NaCl, pH 8.0 Buffer. The Q-Sepharosecolumn helped to separate Collagenase ABC I from Collagenase ABC II inaddition to removing residual DNA impurities and endotoxins. The peakfractions for Collagenase ABC I and ABC II were collected and pooledseparately and stored at −20° C.

In comparison to the methods disclosed in U.S. Pat. No. 7,811,560 inwhich cells were grown in the presence of phytone and glucose, thecurrently claimed method utilizes Veggietone growth media withoutadditional glucose. The currently claimed methods result in thesurprising reduction in the production of clostripain. When proteasesare present in a crude collagenase mixture, skilled artisans must makeuse of protease inhibitors, such as leupeptin, and must performpurification steps in specially designed cold rooms with chilledsolutions. The currently claimed method drastically reduces the amountof clostripain produced, thus eliminating the need for adding proteaseinhibitors, such as leupeptin to the reagents and solutions and furtherallowing a researcher to conduct all purification steps at ambienttemperature.

Example 8 BAEE Assay of Clostripain Activity

Clostripain activity was analyzed using an enzymatic assay that usesN-a-Benzoyl-L-Arginine Ethyl Ester (BAEE) as a substrate. The enzyme wasactivated with 2.5M DTT prior to being mixed with BAEE. The rate of BAEEbreakdown was measured by spectrophotometer at 253 nm, Light path=1 cm.Assay conditions: T=25° C., pH=7.4, 0.75 mM BAEE. One unit of enzyme canhydrolyze 1.0 mole of BAEE per minute. The results are shown in Table 4.The results of the BAEE assay show that clostripain activity wasdrastically reduced in collagenase preparations derived from thosecultures that were grown in Veggietone when compared to collagenasepreparations derived from cultures grown in phytone.

TABLE 4 Growth condition Activity (U/mg) Collagenase I + II from Phytone15.973 Collagenase ABC I + II from bovine materials Batch 1 0.25Collagenase ABC I + II from bovine materials Batch 2 No activityCollagenase ABC I from Veggietone batch 1 No activity Collagenase ABC IIfrom Veggietone batch 1 0.08 Collagenase ABC I from Veggietone batch 2No activity Collagenase ABC II from Veggietone batch 2 No activity

Example 9 Collagenase Assay Method 1: SRC Assay

The soluble rat tail collagen (SRC) assay uses soluble rat tail collagenas a substrate to measure collagenase activity (Mallya S K et al anal.Biochem., 158, 334-345). This assay procedure consists of two majorparts: 1) the enzymatic reaction involving the digestion of rat tailtendon collagen by collagenase and; 2) the subsequent measurement ofliberated peptide fragments/amino acids with ninhydrin reagent.

Method:

Collagenase was incubated for 2.5 hours with soluble rat tail collagen.The extent of collagen breakdown was determined using the Moore andStein (1948) colorimetric ninhydrin method. Amino acids liberated areexpressed as micromoles leucine per milligram collagenase. One unitequals one micromole of L-leucine equivalents from collagen 2.5 hours at25° C. and pH 7.4 under the specified conditions. One net collagenaseunit will solubilize ninhydrin reaction material equivalent to 1nanomole of leucine per minute.

Reagents:

10 mM HCl; 100 mM Tris(hydroxymethyl)aminomethane/20 mM calcium acetate,pH 7.4 at 25° C. (TC assay buffer); Enzyme buffer: 20 mMTris(hydroxymethyl)aminomethane/4 mM calcium acetate, pH 7.4 at 25° C.;Collagen solution (2 mg/mL collagen in 10 mM HCl); 0.5M HCl; Leucinestandard assay solution (1 mM leucine); Rosen buffer: 2.6M sodiumacetate/0.2 mM sodium cyanide/1.1M acetic acid; 3% Ninhydrin in2-methoxyethanol; 50% Isopropanol.

Procedures:

Incubation: Collagenase was incubated with enzyme buffer, HCl, TC Assaybuffer, and Collagen in a 25° C. water bath for 150 minutes.

Quenching: At the end of the incubation period, the reaction tubes wereremoved from the water bath, and 2 mL of 0.5M HCl was added to quenchthe reaction.

Color Development and Reading: A 1 mL aliquot from each of the reactiontubes was transferred into the appropriately-labeled culture tubes. 0.5mL Rosen buffer and 0.5 mL 3% ninhydrin were dispensed into each culturetube. The culture tubes were placed in a boiling water bath for 15minutes, after which the tubes were removed from the boiling water bath,and 3 mL of 50% isopropanol was added. The tubes were allowed to coolfor at least 20 minutes and the absorbances (A₅₇₀) of each culture tubewere read and the readings recorded. The results are shown in Table 5.

Example 10 Collagenase Assay Method 2: GPA Assay

Carbobenzoxy-glycyl-L-prolyl-glycyl-glycyl-L-prolyl-L-alanine (zGPGGPA)is a synthetic substrate for Clostridial collagenase. This substrate isreadily cleaved by Class II collagenases (collagenase ABC II) into thetwo peptides; carbobenzoxy-glycyl-L-prolyl-glycine (zGPG) andglycyl-L-prolyl-L-alanine (GPA). The released free amino group on GPA isreacted with ninhydrin reagent. The optical density of purple ninhydrinreaction solution is measured with spectrometer at 570 nm and comparedwith ninhydrin reaction from collagnease reference standard. The unit ofcollagenase activity was expressed as nmol leu equiv./min. The procedureis based on that of W. Appel (in H. U. Bergmeyer, ed., Methods ofEnzymatic Analysis; New York: Academic Press/Verlag Chemie, 1974).

Solutions:

Appel's tris buffer [0.05M tris (hydroxymethyl) aminomethane/0.1Mcalcium acetate, pH 7.2; substrate solution [2 mg/mL (3.4 mM) zGPGGPA inAppel's tris buffer]; Leucine standard assay solution (1 mM leucine);0.5M HCl; Rosen buffer (2.6M sodium acetate/0.2 mM sodium cyanide/1.05Macetic acid); 3% Ninhydrin; 50% Isopropanol.

Procedure

1.00 mL substrate solution was added into each reaction tube. Theappropriate amount of buffer was added to each reaction tube. All tubeswere placed in a water bath and collagenase was added to each pre-warmedreaction tube. After 10 minutes in the 25° C. water 0.50-mL aliquot wasremoved and transferred to the correspondingly labeled culture tube with0.5 mL HCl.

Color Development and Reading: 0.5 mL Rosen buffer and 0.5 mL 3%ninhydrin were dispensed into each culture tube and the tubes wereplaced in a boiling water bath for 15 minutes. At the end of the boilingperiod, the tubes were removed from the boiling water bath, and 3 mL of50% isopropanol was added to each tube. The tubes were allowed to coolfor at least 20 minutes and the absorbances (A₅₇₀) of each culture tubewere read and recorded. The results are displayed in Table 5.

Example 11 SDS-PAGE, SRC and GPA Analysis of the Post Q ColumnCollagenase

Purified aliquots of collagenase ABC I in tris buffer stored at 4° C.for 0, 7 and 17 days were evaluated for biological activity using theSRC assay and for primary structure integrity using SDS-PAGE, CoomassieBrilliant Blue staining. Purified aliquots of collagenase ABC II in trisbuffer stored at 4° C. for 0, 7 and 17 days were evaluated forbiological activity using the GPA assay and for primary structureintegrity using SDS-PAGE, Coomassie Brilliant Blue staining. The resultsare shown in table 5.

TABLE 5 Stability at 4° C. of the post Q column collagenase ABC I ABC IIdays SRC U/mg SDS-PAGE GPA U/mg SDS-PAGE 0 3327 — 108038 — 7 3298Similar to 0 days 109068 Similar to 0 days 17 3299 Similar to 0 days105023 Similar to 0 days

The results of the SRC, GPA, and SDS-PAGE analyses surprisingly revealthat collagenase remains stable and active in solution even after beingstored for as long as 17 days. There is no activity lost and no visiblecollagenase protein degradation. Such stability and lack of degradationwould not be expected in samples that contained levels of clostripainand other contaminating proteases. Thus, it is clear from thecollagenase and clostripain assays that the currently disclosedfermentation methods utilizing vegetable-based peptone with high saltand no glucose were superior to known methods in reducing clostripainlevels in purified collagenase samples. This result is consistent withthe clostripain test result in example 8.

Example 12 Comparison Between a Collagenase Reference Standard andCollagenase Purified from Medium #4 Culture (Veggitone)

The purified collagenase I and II from M#4 were tested side by side witha collagenase reference standard.

TABLE 6 Test Results on collagenases purified from Veggitone Medium(M#4) Test Results Class I from Class II I + II Test M #4 from M #4 fromM#4 Identity by Same as RS Same as RS Same as RS SDS-PAGE (110-115 kDa)(107-110 kDa) Analysis by Same Retention Same Retention N/A reversephase time as Standard time as Standard HPLC purity > 95% purity > 95%Identity by Conforms to ref Conforms to ref N/A peptide map standardstandard SRC assay: 2888 N/A 2784 specific activity (1967-3327(2441-3759 units/mg) U/mg) GPA assay: N/A 91279 57061 specific activity(81934-119522 (42923-64384 units/mg) U/mg) Clostripain No detectable Nodetectable N/A activity activity activity

The results from Table 6 reveal that the collagenases purified accordingto the methods disclosed herein were comparable to a known referencestandard collagenase with respect to their activity and stability.

The methods of the current invention provide a means for producingessentially clostripain-free preparations of collagenase, thuseliminating the need for using protease inhibitors, such as leupeptin.Furthermore, the elimination of detectable amounts of clostripainactivity from the cultures allows researchers to perform thepurification steps at ambient temperature.

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. All UnitedStates patents and published or unpublished United States patentapplications cited herein are incorporated by reference. All publishedforeign patents and patent applications cited herein are herebyincorporated by reference. All other published references, documents,manuscripts and scientific literature cited herein are herebyincorporated by reference.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. A medium for the fermentation of Clostridium histolyticum comprising:a. a peptone selected from the group consisting of Oxoid VG100 VegetablePeptone made from pea, Oxoid VG200 Vegetable Peptone phosphate broth,BBL Phytone Peptone, and BD Difco Select Phytone; b. yeast extract; andc. less than about 5 g/L glucose; wherein the pH of said medium is7.5-7.9.
 2. The medium of claim 1, containing greater than about 5 g/Lsalt.
 3. The medium of claim 2, containing greater than about 7.5 g/Lsalt.
 4. The medium of claim 2, wherein the salt is one or more ofpotassium phosphate, dipotassium phosphate, sodium phosphate, disodiumphosphate, sodium chloride, potassium chloride, calcium chloride, ormagnesium sulfate.
 5. The medium of claim 1, further comprising one ormore vitamins selected from the group consisting of ferrous sulfate,riboflavin, niacin, calcium pantothenate, pimelic acid, pyridoxinehydrochloride and thiamine hydrochloride.
 6. A medium for thefermentation of Clostridium histolyticum comprising: a. a peptoneselected from the group consisting of Oxoid VG100 Vegetable Peptone madefrom pea, Oxoid VG200 Vegetable Peptone phosphate broth, BBL PhytonePeptone, and BD Difco Select Phytone; b. yeast extract; and c. greaterthan about 5 g/L salt; wherein the pH of said medium is 7.5-7.9.
 7. Themedium of claim 6, containing less than about 5 g/L glucose.
 8. Themedium of claim 6, containing greater than about 7.5 g/L salt.
 9. Themedium of claim 6, wherein the salt is one or more of potassiumphosphate, dipotassium phosphate, sodium phosphate, disodium phosphate,sodium chloride, potassium chloride, calcium chloride, or magnesiumsulfate.
 10. The medium of claim 6, further comprising one or morevitamins selected from the group consisting of ferrous sulfate,riboflavin, niacin, calcium pantothenate, pimelic acid, pyridoxinehydrochloride and thiamine hydrochloride.